The present invention relates to a placental protein and its uses.
References referred to in the text by a number enclosed by parenthesis are listed at the end of the specification.
The goal of pregnancy management is the delivery of a mature, healthy infant, without encountering complications which can adversely affect the well being of both the mother and the newborn. A significant percentage of pregnancies are affected by various disorders. Among these complications are preterm labor and delivery, intrauterine growth retardation and preeclampsia. These conditions negatively impact the outcome of affected pregnancies, at enormous cost both to the patients as well as to the health system.
Placental Protein 13 (PP13) is a protein which was previously isolated from human placental tissue (U.S. Pat. No. 4,500,451 to Bohn, et al., the contents of which are incorporated herein by reference). The protein was characterized by the following parameters: electrophoretic mobility, isoelectric point, sedimentation coefficient, molecular weight determined by ultracentrifugation, molecular weight determined by SDS-PAGE, extinction coefficient and carbohydrate content. The amino acid composition (residues per 100 residues) was determined but not the amino acid sequence.
PP13 was used to develop an assay for the early stage detection of three specific pregnancy-related disorders: intrauterine growth retardation, preeclampsia and preterm delivery (U.S. Pat. No. 5,198,366 to Silberman). Both a radioimmunoassay (RIA) and an enzyme-linked immunoassay (ELISA) are disclosed using labeled PP13 and anti PP13 antiserum, respectively. No farther properties of PP13 are disclosed in the Silberman patent.
It is an object of the present invention to provide a pure PP13 protein.
It is a further object of the present invention to provide a DNA molecule encoding PP13.
It is a still further object of the invention to provide a recombinant method for producing PP13.
Additionally, it is an object of the present invention to provide a diagnostic assay based on PP13 for the early detection of pregnancy complications.
It is another object of the invention to provide immunogenic peptides derived from PP13 which can be used in such a diagnostic assay.
According to one aspect of the present invention, there is provided a protein or polypeptide selected from the group consisting of: (a) Placental Protein 13 (PP13) having the amino acid sequence shown in FIG. 2 (SEQ.ID.NO: 9); (b) a polypeptide having a sequence of amino acids included in PP13 and which binds to antibodies which specifically bind to PP13; (c) a protein or polypeptide of (a) or (b) in which one or more amino acids have been added, deleted or replaced without reducing the ability of the protein or polypeptide to bind antibodies which specifically bind to PP13; and (d) a protein or polypeptide having an amino acid sequence including the amino acid sequence of (a) or (b) or (c).
By another aspect of the present invention, there is provided a DNA molecule encoding the above protein or polypeptide.
According to another aspect of the present invention, there is provided a method of screening for pregnancy-related complications comprising the steps of: (a) providing a serum sample of a pregnant woman; (b) determining the level of PP13 or a peptide derived therefrom in the serum sample. and (c) comparing the determined level with predetermined normal levels for women at the same gestational age, a deviation between the levels being indicative of a pregnancy-related complication.
By one embodiment of the invention, the determination in step (b) is by means of antibodies, preferably monoclonal antibodies, directed against said proteins or polypeptides.
According to yet another aspect of the present invention, there is provided a recombinant method for the production of PP13 comprising inserting said DNA molecule into an expression vector, inserting the expression vector into a host cell, and incubating the host cell under conditions which permit expression of the inserted vector.
The present invention provides for the first time the full amino acid sequence of PP13, as well as its fall cDNA sequence. This information can be utilized in a number of applications. For example, modified PP13 protein homologues and analogues can be produced in which one or more amino acids have been added, deleted or replaced, the modified protein typically retaining 75% homology with PP13. Methods for modifying the amino acid sequence of a protein whose fall sequence is known are well known in the art, and include e.g. chemical synthesis, controlled mutagenesis and recombinant methods. Such modified proteins may have superior properties over the natural PP13 in various applications, such as superior immunogenicity or immuno-specificity (e.g. the modified protein may be devoid of immune epitopes common with other proteins) for use in an immunoassay for the early detection of pregnancy-related disorders as described in Silberman.
Furthermore, peptide fragments may be prepared from PP13 and such peptides may be modified as described above with respect to the full protein. These peptides may also be used in various applications. For example, it is well known that immunogenic proteins have specific amino acid sequences or epitopes which induce the immune system to mount an immune response to the protein. The above peptides may be tested for the presence of an epitope of PP13 so as to identify the epitope(s). A peptide containing an epitope may then be used in an immunoassay for pregnancy disorders. A number of PP13 derived peptides are disclosed below.
The pure PP13 protein or a derived peptide may be used to prepare antibodies to PP13. Either polyclonal or monoclonal antibodies may be produced by standard methods well known to the skilled artisan.
Both the antibodies as well as the proteins and peptides may be used to prepare diagnostic or screening assays for the detection of pregnancy-related complications such as intrauterine growth retardation, preterm delivery and preeclampsia. Examples of such assays are detailed in Silberman, the contents of which are incorporated herein by reference, and include radioimmunoassays (RIA) and enzyme-linked immunoassays (ELISA). In general, such an assay will include the steps of obtaining a serum sample of a pregnant woman, determining the level of PP13 or of a derived peptide in the serum sample by the immunoassay, and comparing the determined level with pre-determined normal levels for women at the same gestational age. A statistically significant deviation between the levels will be indicative of a pregnancy-related complication.
As mentioned above, the full cDNA of PP13 is disclosed here for the first time. Since the full amino acid sequence of PP13 is also disclosed, various DNA molecules encoding PP13 may be prepared due to the degeneracy of the genetic code. In addition, DNA molecules capable of hybridizing to these DNA molecules under stringent conditions may also be prepared. The DNA molecules may be used in a recombinant method for the production of PP13. Such methods are well known in the art and usually involve inserting the DNA molecule into an expression vector such as a plasmid, phage or viral DNA. The expression vector is then inserted into a compatible host cell such as bacterial cells, or eukaryotic cells such as yeast, plant, mammalian or insect cells. The host cell is incubated under conditions which induce expression of the inserted vector, thereby producing PP13.
For example, the DNA encoding PP13 can be inserted into an expression vector under the control of an inducible promotor such as the LacZ promoter, T7 or T4 polymerase promoter, heatshock promoters, etc. One example of an expression vector is the pQE expression vector (QIAGEN. The pQE vector provides high level expression of proteins containing a 6*His affinity tag in E. coli. The pQE contains a regulatable promoter consisting of the E. coli phage T5 promoter and two lac operator sequences. The vector is then inserted into a competent M15 [PREP4] E. coli strain (Villarejo and Zabin. 1974). The M15 host cell contains multiple copies of the plasmid pREPA which carries the lacl gene encoding the lac repressor. The host cell is incubated with IPTG which rapidly induces expression of the inserted vector, thereby producing PP13. Many other systems may also be used for PP13 expression, as is well known to the skilled artisan.
A kit for diagnosing pregnancy-related complications may be produced based on the present invention. Such a kit, for example, may comprise the following components: (1) antibodies capable of specifically binding PP-13; (2) labeled PP-13, for example by a radioactive, fluorescent or enzyme marker; (3) PP-13 standard solutions at known concentrations; and (4) means for detecting the signal produced in the assay. Such means could be, for example, antiserum raised against the PP-13-binding antibodies.